Studies on the Novel Enzymatie Reaction Mechanisms Taking into Account the Enzyme Fluctuations

为了阐明酶的构象波动在催化机理中的重要意义，研究了不同丝氨酸蛋白酶催化水解的速率常数与介质粘度的关系。对硝基苯胺对α-胰凝乳酶的酰化作用不受介质粘度的影响，而对硝基苯酯对α-胰凝乳酶的酰化作用随介质粘度的增加而降低。在β-胰蛋白酶催化的水解过程中也观察到这种趋势。对于这些酶，酶的构象效应存在于四面体中间体的形成中，而其分解几乎不受构象效应的影响。另一方面，除了对硝基苯基酯对嘉士伯枯草杆菌的酰化外，对硝基苯胺的酰化也受到介质粘度的影响。这表明（ES）_T的形成和随后的分解都是由酶的构象变化辅助的。这些结果表明，虽然丝氨酸蛋白酶具有相同的催化机制，但在催化过程中构象效应存在一定差异。为了将该方法扩展到蛋白酶以外的酶，从面包酵母细胞中分离纯化了几种新的还原酶，它们催化硝基烯和α，β-不饱和酮的碳-碳双键的不对称还原。

In order to clarify the significance of conformational fluctuation of an enzyme in the catalytic mechanism, the dependence of rate constants for hydrolyses catalyzed by various serine proteases on medium viscosity were investigated. Acylation of α-chymotrypsin by a p-nitroanilides is hardly affected by medium viscosity, while that by a p-nitrophenyl ester decreases with increase in medium viscosity. This tendency is also observed in β-trypsin-catalyzed hydrolyses. For these enzymes, the conformational effect of the enzyme exists in formation of a tetrahedral intermedite, whereas its breakdown is almost free from the conformational effect. On the other hand, in addition to the acylation of subtilisin Carlsberg by p-nitrophenyl esters, that by p-nitroanilides is also influenced by medium viscosity. This suggests that both formation of (ES)_T and following breakdown of it are assisted by the conformational change of the enzyme. These results reveal that, although serine proteases have the same catalytic mechanism each other, there have some differences in the conformational effects during the catalytic processes.In order to extend this method to the enzymes other than proteases, several novel reductases were isolated and purified from the cells of baker's yeast, which catalyze the asymmetric reduction of a carbon-carbon double bond in nitroalkenes and α,β-unsaturated ketones.